Development And Function Of Mammalian Chemosensory Syste

分子神经科学的研究目标是确定哺乳动物化学感受系统的发展和功能的分子机制。过去一年的研究工作一直致力于建立苦味受体的功能测定，并鉴定和表征在味觉受体细胞中选择性表达的新基因。 为了表征苦味受体的人T2 R家族成员的功能特性，我们已经开发了这些受体的体外重建测定。对于该测定，用23种人T2 R中的每一种构建杆状病毒表达载体。从感染这些杆状病毒的昆虫细胞中分离的细胞膜的Western分析表明，膜高度富集苦味受体。使用这些膜组分，我们能够通过添加纯化的G蛋白来功能性地重建受体活性。我们目前正在筛选hT 2 R家族的孤儿成员与一组60个苦味化合物，以揭示配体-受体相互作用。到目前为止，我们已经确定了四种新的受体-配体相互作用，包括马兜铃酸、地那铵和氯喹的受体。使用这些定义的受体/配体对，我们现在正在进行定量评估的配体结合特性和G蛋白的选择性，这些受体。这些研究将为深入了解苦味受体的特异性及其激活的信号转导途径提供帮助。 在过去的一年中，我们还表征了一种新的2B家族G蛋白偶联受体GPR 113，它特异性地表达于味觉受体细胞中。2B家族是G蛋白偶联受体的一个新的亚群，由大约30个成员组成，其中只有三个成员的功能已被确定。GPR 113与已知的甜味和苦味受体显示出很小的序列同源性，并且是其类别中第一个显示出在味觉受体细胞亚群中选择性表达的。GPR 113的长胞外结构域包含一个肽结合结构域，其特征性地见于充分研究的肽激素结合G蛋白偶联受体中，表明GPR 113的一个配体是肽。作为一种假定的肽受体，GPR 113可以识别内源性神经肽，例如，可以调节味觉敏感性。或者，GPR 113可能作为一个味觉受体的许多摄入的肽之一，已知引发味觉。

The research goals of the Section of Molecular Neuroscience are to define the molecular mechanisms underlying the development and function of mammalian chemosensory systems. Research efforts this past year have been directed towards establishing functional assays for bitter taste receptors and identifying and characterizing novel genes selectively expressed in taste receptor cells. To characterize the functional properties of members of the human T2R family of bitter receptors, we have developed an in vitro reconstitution assay for these receptors. For this assay, baculoviral expression vectors were constructed with each of the 23 human T2Rs. Western analyses with cellular membranes isolated from insect cells infected with these baculoviruses indicate that the membranes are highly enriched in bitter receptors. Using these membrane fractions, we are able to functionally reconstitute receptor activity by the addition of purified G proteins. We are currently screening orphan members of the hT2R family with a panel of 60 bitter-tasting compounds to uncover ligand-receptor interactions. Thus far, we have identified four novel receptor-ligand interactions including receptors for the bitter-tasting compounds aristolochic acid, denatonium, and chloroquine. Using these defined receptor/ligand pairs, we are now performing quantitative assessments of the ligand binding properties and the G protein selectivities of these receptors. This studies will provide insight into the both the specificities of bitter receptors and the signal transduction pathways they activate. This past year we also characterized a novel family 2B G-protein-coupled receptor, GPR113, that is specifically expressed in taste receptor cells. The 2B family, a new subgroup of G-protein-coupled receptors, consists of about 30 members of which only three have been functionally characterized. GPR113 displays little sequence homology to known sweet and bitter taste receptors and is the first of its class shown to be selectively expressed in a subset of taste receptor cells. The long extracellular domain of GPR113 contains a hormone-binding domain, characteristically seen in the well-studied peptide hormone binding G-protein-coupled receptors, suggesting one ligand for GPR113 is a peptide. As a putative peptide receptor, GPR113 could function to recognize an endogenous neuropeptide that could, for example, act to modulate taste sensitivities. Alternatively, GPR113 might act as a taste receptor for one of the many ingested peptides that are known to elicit taste sensations.